This project focuses on the transcriptional control of the prodynorphin gene, which codes for the dynorphin family of opioid peptides. Peripheral inflammation greatly increases dynorphin gene expression in spinal cord neurons where peptides can modulate chronic pain. Transient transfection using in vitro cell lines indicate that the dynorphin gene is turned on by the cyclic AMP (cAMP) second messenger system. A dual feed-forward stimulatory transcription scheme is proposed in which members of the CREB/ATF and Fos/Jun families positively transactivate the dynorphin gene. The results imply that a transmitter in primary afferent neurons is linked to stimulation of adenyl cyclase in second order neurons and that the gene regulatory and neuronal excitability changes which accompany chronic pain may be engendered or maintained by cAMP-dependent phosphorylation. Additional functional assessments of the DYNCRE3 sequence were performed with a series of DYNCRE3 mutations. The data demonstrate that (a) an intact core heptanucleotide region is required for optimal basal and inducible expression, (b) the immediate two flanking bases are of minimal importance, (c) nucleotides further from the core influence activity but less so than core mutations, and (d) under different conditions of stimulation the DYNCRE3 element exhibits characteristics of an AP-1 element or a CRE element. These data indicate that the DYNCRE3 element provides a more flexible (and complex) transcriptional responsiveness than either of the two consensus sequences. Based on the above in vitro observations we have begun corresponding experiments using the rat peripheral inflammation model. These include examination of Jun and CREB proteins and their phosphorylation status and assessment of the effects of modulation of the cAMP system on nociceptive sensitivity and neuropeptide gene.